1. Field of the Invention
The present invention relates to a process for producing hydrogen with use of a microorganism possessing the hydrogenase gene and having the lactic acid and succinic acid biogenetic pathways inactivated in its anaerobic metabolic pathways. The hydrogen generated by the process of the present invention is usable as a fuel for fuel cells.
2. Description of the Related Art
Hydrogen has an increased attention as an ultimate clean energy source, which, unlike fossil fuels, does not liberate any environmentally controversial substances through combustion, such as carbon dioxide gas, sulfur oxide and the like. Hydrogen can deliver a heat quantity per unit mass three times or more greater than petroleum oils, and when supplied to fuel cells, can be converted into electric and thermal energies with a high degree of efficiency.
As a conventional chemical process for production of hydrogen, for example, there have been proposed several technologies, inclusive of the thermal-cracking or steam reforming process of natural gas or naphtha. These production technologies need the reactions conditions of high temperature and high pressure, while they yield the synthesis gas containing CO (carbon monoxide), and such gas, on the occasion of utilization in fuel cells, is consequently required to be freed of CO to circumvent the problem of poisoning of fuel-cell electrode catalysts. However, the removal of CO is technically-difficult and cannot be said to be easy.
On the other hand, the method of biological hydrogen generation by microorganisms can be conducted into practice under the reaction conditions of ambient temperature and atmospheric pressure, and the generated gas does not need the removal of CO, because it does not contain CO.
From described standpoints, the method of biological hydrogen generation with use of microorganisms is attracting enhanced attention as a more preferred means of supplying a fuel intended for use in fuel cells.
The method of biological hydrogen generation is roughly classified into the two methods: method using a photosynthetic microorganism and the method with use of a non-photosynthetic microorganism (mainly an anaerobic microorganism).
The former method, although it uses the energy of light for generation of hydrogen, needs a vast light capturing surface area because of its low utilization efficiency of the energy of light, and encounters many unsolved problems, such as the expensive cost investment for the hydrogen production facilities and difficulties in securing its maintenance administration, thus having still been considered not to reach the stage of commercialization.
With reference to the latter method, there have been known the various metabolic pathways being responsible for generating hydrogen by the anaerobic microorganisms. Such metabolic pathways include, for example, the pathway of generating hydrogen during the step of decomposition of glucose to pyruvic acid; the pathway of generating hydrogen during the step of production of acetic acid from pyruvic acid via acetyl CoA; or the pathway of generating hydrogen directly from formic acid originating from pyruvic acid, and the like. The hydrogen generation, which is conjugated with reoxidation of NADH and the like in the step of decomposition of glucose to pyruvic acid or in the step of production of acetic acid from pyruvic acid via acetyl CoA, is frequently found to take place in obligetory anaerobic microorganisms. The pathway of generating hydrogen directly from formic acid originating from pyruvic acid is working, as a formic acid hydrogen lyase system (hereinafter, referred to briefly as “FHL system”), in lots of different microorganisms including facultative anaerobic microorganisms, and the FHL system has been investigated extensively in the species Escherichia coli. The generation of hydrogen from glucose by facultative anaerobic bacteria, which may be typified by Escherichia coli, is considered to proceed through the successive steps of producing two molecules of pyruvic acid generated from one molecule of glucose and producing two molecules each of acetyl CoA and formic acid from one mole of glucose through cleavage of pyruvic acid into acetyl CoA and formic acid, followed by decomposition of formic acid to hydrogen and carbon dioxide. Accordingly, the theoretical yield of the hydrogen generation from one molecule of glucose by such facultative anaerobic bacteria is regarded as 2 molecules.
There have been known so far several processes for producing hydrogen from glucose used as a substrate through the FHL system, and a research paper was published on the process for producing hydrogen by the method of inactivation of the lactic acid biogenetic pathway in Escherichia coli. 
Koji Sode et al. in Biohydrogen II, 2001, p. 195-204 disclosed the process for producing hydrogen from glucose using the microorganism having its lactic acid generation pathway inactivated. However, it is noticed that the hydrogen yield was hardly improved in spite of the fact that the lactic acid generation pathway was inactivated, as may be reflected by the description mentioning that the hydrogen yield from glucose was realized merely in the range of as low as about 22% to 24% to the theoretical yield, while at the same time, the rate of hydrogen generation was not improved, as well.
The Official Gazette of WO 2004/074495 discloses the method for providing a microorganism capable of generating hydrogen, which method comprises culturing a microorganism under aerobic conditions and culturing the resultant microbial cells under anaerobic conditions to impart the hydrogen generating capability to the microorganism. However, the publication does not make mention of improvements both in rate of hydrogen generation and hydrogen yield to be attained through gene modification.
K. Y. ALAM et al., in J. Bacteriol., 1989, Vol. 171, p. 6213-6217, reported the generation balance of organic acids and the like in relation to various substrates having different levels of oxidation-reduction potential. This literature reference describes that there was obtained the microorganism capable of suppressing the generation of lactic acid and succinic acid through inactivation of the lactate dehydrogenase gene (hereinafter also referred to briefly as ldhA) and also the fumarate reductase gene (hereinafter also referred to as frdABCD), whereas it does in no way describe anything about the hydrogen generation and hydrogen yield, although it gives the description about the oxidation-reduction potential balance of the resultant products. The fumarate reductase is considered to be composed of four subunits of four kinds of the proteins encoded by the four genes, frdA, frdB, frdC, and frdD, to elicit the enzymatic activity as a composite enzyme, whereas the said literature reference does neither describe how the fumarate reductase (FRD) was inactivated nor indicate which subunit was inactivated.
The said literature reference, in Table 3 (page 6215), presents the compositions of various organic acids and the like generated from different types of saccharides used, and also describes the data demonstrating that the mutant strains, which had the lactate dehydrogenase gene and fumarate reductase gene frd inactivated, exhibited almost no change in the generation amount of formic acid supposedly associated with the hydrogen generation, as compared with the wild-type. The document does neither suggest the procedural technique of improving the yield and rate of hydrogen generation from saccharides nor infer the relevant phenomena with increasing hydrogen production.
Biotechnol Lett., 2001, 23, p537-541, discloses that the hydrogen yield was greatly increased by inactivating the anaerobic metabolism pathway with use of a mutagenic substance. However, the report does neither specify the inactivated gene nor describe anything about succinic acid.
The official Gazette of PCT/JP2004/002092 discloses a process for producing hydrogen with a highly enhanced efficiency, but the method poses restriction on the conditions for culturing microbial cells in the presence of formic acid, and does not teach any improvement of the hydrogen yield to be attained by the microorganism through genetic recombination.
With reference to the hydrogen yield from an organic substrate under anaerobic conditions, for example, the theoretical hydrogen yield is 2 molecules per molecule of glucose. In practice, however, it is difficult to generate hydrogen in the theoretical yield, because discharge of the reductive power which is the basic working principle of energy source for the hydrogen generation by anaerobic microorganisms, is used in other forms different from the hydrogen generation. In addition, the methods for producing hydrogen with use of a microorganism suffer from the disadvantage that the rate of hydrogen generation is slow.